Flavivirus fusion inhibitors

ABSTRACT

The present invention relates to peptides and methods of inhibiting fusion between the virion envelope of Flaviviruses and membranes of the target cell, the process that delivers the viral genome into the cell cytoplasm. The invention provides for methods which employ peptides or peptide derivatives to inhibit Flavivirus:cell fusion. The present invention is based in part on the discovery that E 1  envelope glycoprotein of hepaciviruses and E 2  envelope glycoprotein of pestivirus have previously undescribed structures, truncated class II fusion proteins. The present invention provides peptides and methods of treatment and prophylaxis of diseases induced by Flaviviruses.

This Application claims the Benefit of U.S. Provisional Application Ser.No. 60/424,746, filed Nov. 8, 2002, which is incorporated by reference,in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to peptides and methods of inhibiting cellinfection and/or virion:cell fusion by members of the Flaviviridaefamily.

2. BACKGROUND OF THE INVENTION

2.1. Entry of enveloped animal viruses requires fusion between the viralmembrane and a cellular membrane, either the plasma membrane or aninternal membrane. Class I fusion proteins possess a “fusion peptide” ator near the amino terminus, a pair of extended α helices and, generally,a cluster of aromatic amino acids proximal to a hydrophobictransmembrane anchoring domain (Carr and Kim, 1993; Suarez et al., 2000;Wilson, Skehel, and Wiley, 1981). Several otherwise disparate viruses,including orthomyxoviruses, paramyxoviruses, retroviruses, arenaviruses,and filoviruses encode class I fusion proteins varying in length andsequence, but highly similar in overall structure (Gallaher, 1996;Gallaher et al., 1989). X-ray crystallography of the E glycoprotein(E-protein) of tick-borne encephalitis virus (TBEV), a member of thegenus flavivirus of the Flaviviridae family, revealed a structure forthis fusion protein distinct from other fusion proteins (Rey et al.,1995). E-protein possesses an internal fusion peptide stabilized bydicysteine linkages and three domains (1-111) comprised mostly ofantiparallel β sheets. In the slightly curved rod-like configuration ofthe E-protein present in the virion, the fusion peptide is located atthe tip of domain II, the furthest point distal from the C-terminaltransmembrane anchor. Examination by Lescar and coworkers (2001) of E1,the fusion protein of the Togavirus Semliki Forest virus (SFV), revealeda remarkable fit to the scaffold of TBEV E. Recently, the E-glycoproteinof dengue virus, a medically important flavivirus, was also shown tohave a class II structure (Kuhn et al., 2002).

2.2. The Flaviviridae family consists of three genera, flaviviruses,hepaciviruses and pestiviruses. In the United States alone, 4 millionpeople are infected with a member of the hepacivirus genus, hepatitis Cvirus (HCV). This is four times the number infected by HIV. Each year inthe US, 30-50,000 new HCV infections occur, and about 15-20,000 peopledie. These numbers are expected to increase dramatically. The infectionis spread primarily through needle sharing among drug users, althoughthere is some risk from accidental needle sticks, blood products before1992, chronic blood dialysis, and frequent sexual contact. Currenttreatments for HCV using ribavirin and interferon cost $8,000 to $20,000per year, and help about half of patient only partly. End stage HCVdisease is the most frequent indication for liver transplants and thiscosts $250,000 to $300,000. Better drugs to treat HCV infection and aneffective vaccine to prevent HCV infection are urgently needed. Membersof the flavivirus genus, dengue virus, Japanese encephalitis virus,yellow fever virus, and West Nile virus, cause important human diseasesworld-wide. Pestiviruses, such as bovine viral diarrhea virus and borderdisease virus, cause significant veterinary illnesses.

3. SUMMARY OF THE INVENTION

Based on sequence similarities, it is likely that the E glycoproteins ofother members of the flavivirus genus within the family Flaviviridae,including West Nile virus, are also class II fusion proteins. Analysespresented herein indicate that glycoproteins of viruses from members ofthe other two genera of the Flaviviridae family, hepaciviruses andpestiviruses, have previously undescribed structures. The envelopeglycoprotein E1 of hepatitis C virus, a hepacivirus, and the envelopeglycoprotein E2 of pestiviruses have novel structures, resembling atruncated version of a class II fusion protein. No viral protein haspreviously been identified with this structure. Our observations wereunexpected and contrast with published studies. Hepatitis C virusencodes two envelope glycoproteins, E1 (gp35) and E2 (gp70), both withC-terminal transmembrane anchor domains. Prior studies indicated thatanother HCV protein, E2, has a class II structure. The structuraldeterminations of the hepacivirus and pestivirus fusion proteins allowthe identification of several heretofore unknown features of Flavivirusfusion proteins for drug and vaccine development.

Thus, the instant invention teaches that HCV envelope glycoprotein E1has a previously unknown structure, a truncated class II fusion protein.This structure identifies regions of HCV E1 and other class II viralfusion proteins important for virus:cell fusion. This invention alsoteaches that peptides can be designed to inhibit viruses, including HCVand other members of the Flaviviridae family, that have fusion peptideswith a class II structure.

Structural features of Flavivirus envelope glycoproteins identifiedherein provide surprising guidance for the development of vaccinesand/or drugs to prevent or treat Flavivirus infections. Prior to theavailability of X-ray structural data (Wild, Greenwell, and Matthews,1993; Wild et al., 1994), several potent HIV-1 TM inhibitors weredeveloped based on the Gallaher HIV-1 TM fusion protein model (Gallaheret al., 1989). DP178 (T20) peptide (FIG. 5A) has been shown tosubstantially reduce HIV-1 load in AIDS patients in preliminary resultsfrom phase III clinical trials. (Hoffman-La Roche and Trimeris, 2002).Peptide drugs should be relatively easy to develop, based on ourstructures. Once an effective peptide inhibitor is described anon-peptide drug can be developed.

More specifically, the present invention provides for methods ofinhibiting viral infection by Flaviviruses and/or fusion between thevirion envelope of Flaviviruses and membranes of the target cell (theprocess that delivers the viral genome into the cell cytoplasm). Theinvention is related to the discovery, as described herein, thathepacivirus envelope glycoprotein E1 and pestivirus E2 glycoprotein havenovel structures. The invention provides for methods that employpeptides or peptide derivatives to inhibit Flavivirus:cell fusion. Thepresent invention provides for methods of treatment and prophylaxis ofdiseases induced by Flaviviruses.

Various embodiments of the instant invention provide for pharmaceuticalcompositions comprising one or more peptides selected from one or moreof the following groups.

-   A) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID    NO:36;-   B) Peptides homologous to any one of SEQ ID NO:1 to SEQ ID NO:36,    except that they are from a different flavivirus.-   C) Peptides that are functionally equivalent to any one of SEQ ID    NO:1 to SEQ ID NO:36, wherein the functionally equivalent peptide is    identical to at least one of SEQ ID NO:1 to SEQ ID NO:36 except that    one or more amino acid residues has been substituted with a    homologous amino acid, resulting in a functionally silent change, or    one or more amino acids has been deleted.

Various aspects of this embodiment of the invention provide forcompositions that comprise one or more peptides selected from thefollowing.

-   A) Peptides having the amino acid sequence one or more of SEQ ID    NO:1 to SEQ ID NO:36, wherein the N-terminal “Xaa” is an amino group    and the C-terminal “Xaa” is a carboxyl group.-   B) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID    NO:36, wherein the N-terminal “Xaa” is not an amino group and/or the    C-terminal “Xaa” is not a carboxyl group, wherein the N-terminal    “Xaa” is selected from the group consisting of: an acetyl group, a    hydrophobic group, carbobenzoxyl group, dansyl group, a    t-butyloxycarbonyl group, or a macromolecular carrier group, and/or    wherein the C-terminal “Xaa” is selected from the group consisting    of an amido group, a hydrophobic group, t-butyloxycarbonyl group or    a macromolecular group.-   C) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID    NO:36 except that at least one bond linking adjacent amino acid    residues is a non-peptide bond.-   D) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID    NO:36, except that at least one amino acid residue is in the    D-isomer configuration.-   E) Peptides as in groups “A)” or “B)” except that at least one amino    acid has been substituted for by a different amino acid (whether a    conservative or non-conservative change).-   F) Peptides that are a functional fragment of a peptide as set out    in any of groups “A)” to “E)”, above, where the peptides have at    least 3 contiguous nucleotides of any one of SEQ ID NO:1 to SEQ ID    NO:36.

The instant invention also provides for substantially purifiedantibodies that specifically react with one or more of the peptidesdescribed above.

The instant invention also provides for methods for treating orpreventing viral infections in an animal where the method comprisesadministering to an animal or human peptides and/or antibodies asdescribed above.

3.1. Abbreviations

-   HIV—human immunodeficiency virus-   TBEV—tick-borne encephalitis virus-   DV—dengue virus-   WNV—West Nile virus-   HCV—hepatitis C virus-   GBV—hepatitis GB virus-   CSFV—classical swine fever virus-   BVDV—bovine viral diarrhea virus-   BD—border disease virus-   HSA—human serum albumen

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Alignments of tick-borne encephalitis virus E, hepatitis C virusE1 and classical swine fever virus E2 glycoproteins. Panel A: Aminoacids are numbered from the beginning of the TBEV, HCV and CSFVpolyproteins in this and subsequent figures. Bracketed HCV insertsequences are wrapped and do not represent an alignment comparison.“(:)” refers to identical amino acids. “(.)” refers to chemicallysimilar amino acids. Panel B: Linear arrangement of the domain structureof TBEV E as determined by Rey et al. (1995). Regions of significantsequence similarities to TBEV E in HCV E1 and E2 and CSFV E2 asdetermined by the PRSS3 sequence alignment program are indicated.Probabilities (p-values) are based on 1000 shuffles.

FIG. 2. Structures of hepacivirus E1 and pestivirus E2 glycoproteins.Panel A. Structure of TBEV E as determined by Rey et al. (1995) is shownschematically (traced from a RasMac molecular visualization softwarerendering). Panel B: Structure of HCV E1. HCV E1 sequences withsimilarity to TBEV E sequences are enclosed in quotation marks. Panel C:Structure of CSFV E2.

FIG. 3. Alignments of the precursor of tick-borne encephalitis virussmall membrane protein, prM, and classical swine fever virus E1. PanelA: alignments were constructed as detailed in the text. Panel B: Lineararrangement of TBEV prM and CSFV E1 with a region of sequence similaritydetermined by the PPSS3 algorithm indicted.

FIG. 4. Common order of proteins in Flaviviridae polyproteins. Proteinsor portions of proteins with similar functions are located in similarlocations along the polyproteins of members of the Flaviviridae.Hydrophobic domains were predicted using TMpred.

FIG. 5. Comparison of human immunodeficiency virus transmembraneglycoprotein (TM) with hepatitis C virus envelope glycoprotein 1 (E1).Panel A: an updated structure of HIV-1 TM from Gallaher et al. (1989)with structural motifs indicated in rainbow order. Amino acids arenumbered from the beginning of the Env polyprotein. HIV-1 TM istruncated after the transmembrane domain. The precise ends of the TM N-and C-helices are unclear because of conflicting structural data. Noattempt was made to align the N- and C-helices, although points ofcontact are solved in the coiled oil formations. Positions of knownneutralizing epitopes on TM are indicated, as well as sequencescorresponding to peptides CS3 and DP178 (T20) (Qureshi et al., 1990;Wild et al., 1994) that inhibit HIV-1 infectivity. Panel B: Structure ofHCV E1 with motifs that are shared with HIV-1 TM. Boxed arrows arepredicted beta sheet structures that are similar to the indicated βsheets of TBEV E. Predicted a helical structures are outlined. Arrowsdenote directions that the HCV E1 structure could fold in threedimensions.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of inhibiting Flavivirusinfection that comprises inhibiting the fusion between the virionenvelope and a cell membrane, the process that delivers the viral genomeinto the cell cytoplasm. For purposes of clarity of disclosure, and notby way of limitation, the description of the present invention will bedivided into the following subsections:

-   (i) peptides of the invention-   (ii) utility of the invention    5.1. Peptides of the Invention

Any peptide or protein which inhibits the fusion between the Flavivirusvirion envelope and a cell membrane, including those of Flaviviruseswhich infect human as well as nonhuman hosts, may be used according tothe invention. In various embodiments of the invention, these inhibitorsmay include, but are not limited to peptides related to severalmembrane-interactive domains of Flavivirus fusion proteins.

Flavivirus inhibitory peptides are, according to the instant invention,identical or homologous to the amino acid sequences HCV FusionInhibitory Protein 1, X-YQVRNSSGLYHVTNDCPNSSIVYEAADAIL-Z (SEQ ID NO:1);HCV Fusion Inhibitory Protein 2, X-CSALYWVGDLCGSVFLVGQLFTFSPRRHWTTQDC-Z(SEQ ID NO:2); HCV Fusion Inhibitory Protein 3,X-SPRRHWTTQDCNCSIYPGHITGHRMAWDMMWSPT-Z (SEQ ID NO:3); or HCV FusionInhibitory Protein 4, X-MMMNWSPTAALLRIPQAIMDMIAGAHWGVLAGIKYFSMVGNW-Z(SEQ ID NO:4), or portions thereof or, alternatively, to a homologouspeptide sequence associated with another Flavivirus, including, but notlimited to, HGB, DV, JEV, YFV, WNV, CSFV, BVDV, or BDV as provided belowin tables 1 through 4.

As used herein the term “homologous peptide” preferably refers tosimilar peptides from other strains of a given virus or, alternativelyfrom related viruses.

As used herein the term “similar peptides” refers to those peptideshaving at least 70% identical or chemically similar amino acids. Morepreferably, it refers to peptides having 75%, 80%, 85%, 90%, 95%, orgreater identical and/or chemically equivalent amino acid resides.

As used herein the terms “portion thereof” refers to the peptideresulting from the removal of from one or more amino acids from eitheror both ends of the listed peptide, i.e. a truncated peptide. The numberof amino acids removed may vary from 1-10 so long as the remainingfragment is “functional”. As defined herein the term “functionalfragment” refers to a fragment capable of inhibiting virus:cell fusion,inhibiting viral infectivity, capable of eliciting an antibody capableof recognizing and specifically binding to non-truncated peptide and/orinterfering with Flavivirus envelope protein-mediated cell infection.TABLE 1 Flavivirus fusion inhibitory peptide 1 PROTEIN SEQUENCE HCV E1X-YQVRNSSGLYHVTNDCPNSSIVYEAADAIL-Z (SEQ ID NO:1) HGB E1X-RVTDPDTNTTILTNCCQRNQVIYCSPSTCL-Z (SEQ ID NO:5) DV EX-RDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDF-Z (SEQ ID NO:6) JEV EX-RDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDV-Z (SEQ ID NO:7) YFV EX-RDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDI-Z (SEQ ID NO:8) WNV EX-RDFLEGVSGATWVDLVLEGDSCVTIMSKDKPTIDV-Z (SEQ ID NO:9) CSFV E2X-GQLACKEDYRYAAISSTNEIGLLGAGGLTTTWKEYN-Z (SEQ ID NO:10) BVDV E2X-GHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYS-Z (SEQ ID NO:11) BDV E2X-GEFACREDHRYALAKTKEIGPLGAESLTTTWTDYQ-Z (SEQ ID NO:12)

TABLE 2 Flavivirus fusion inhibitory peptide 2 PROTEIN SEQUENCE HCV E1X-CSALYWVGDLCGSVFLVGQLFTFSPRRHWTTQDC-Z (SEQ ID NO:2) HGB E1X-TCDALDIGELCGACVLVGDWLVRHWLIHIDLNET-Z (SEQ ID NO:13) DV EX-KRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTC-Z (SEQ ID NO:14) JEV EX-SSYVCKQGFTDRGWWGNGCGLFGKGSIDTCAKFSC-Z (SEQ ID NO:15) YFV EX-GDNACKRTYSDRGWGNGCGLFGKGSIVACAKFTC-Z (SEQ ID NO:16) WNV EX-PAFVCRQGVVDRGWGNGCGLFGKGSIDTCAKFAC-Z (SEQ ID NO:17) CSFV E2X-KGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPT-Z (SEQ ID NO:18) BVDV E2X-RGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMD-Z (SEQ ID NO:19) BDV E2X-RGKYNATLLNGSAFQLVCPYEWTGRVECTTISKS-Z (SEQ ID NO:20)

TABLE 3 Flavivirus fusion inhibitory peptide 3 PROTEIN SEQUENCE HCV E1X-SPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPT-Z (SEQ ID NO:3) HGB E2X-IHIDLNETGTCYLEVPTGIDPGFLGFIGWMAGKVEA-Z (SEQ ID NO:21) DV EX-MVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNW-Z (SEQ ID NO:22) JEV EX-FYVMTVGSKSFLVHREWFHDLALPWTSPSSTAWRNR-Z (SEQ ID NO:23) YFV EX-SYIAEMETESWIVDRQWAQDLTLPWQSGSGGVWREM-Z (SEQ ID NO:24) WNV EX-YYVMTVGTKTFLVHREWFMDLNLPWSSAGSTVWRNR-Z (SEQ ID NO:25) CSFV E2X-TLRTEVVKTFRRDKPFPHRMDAVTTTVENEDLFY-Z (SEQ ID NO:26) BVDV E2X-TLATEVVKIYKRTKRFRSGLVATHTTIYEEDLYH-Z (SEQ ID NO:27) BDV E2X-TLATTVVRTYRRSKPFPHRQGAITQKNLGEDLH-Z (SEQ ID NO:28)

TABLE 4 Flavivirus fusion inhibitory peptide 4 PROTEIN SEQUENCE HCV E1X-MMMNWSPTAALLRIPQAIMDMIAGAHWGVLAGIKYFSMVGNW-Z (SEQ ID NO:4) HGB E1X-WMAGKVEAVIFLTKLASQVPYAIATMFSSVHYLAVGALIYYS-Z (SEQ ID NO:29) DV EX-MAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSW-Z (SEQ ID N0:30) JEV EX-LAALGDTAWDFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMSW-Z (SEQ ID NO:31) YFV EX-LAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNW-Z (SEQ ID NO:32) WNV EX-LAALGDTAWDFGSVGGVFTSVGKAVHQVFGGAFRSLFGGMSW-Z (SEQ ID NO:33) CSFV E2X-QQYMLKGEYQYWFDLDVTDRHSDYFAEFVVLVVVALLGGRYI-Z (SEQ ID NO:34) BVDV E2X-QQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYV-Z (SEQ ID NO:35) BDV E2X-QQYMLKGQYQYWFDLEVISSTHQIDLTEFIMLAVVALLGGRYV-Z (SEQ ID NO:36)

According to the instant invention peptides related to the Flavivirusfusion inhibitory peptides (FIP) preferably comprise at least threecontiguous residues of the FIP peptides, or a homologous peptide, morepreferably they comprise 4, 5, 6, or 7 contiguous residues. Even morepreferably they comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguousresidues, and most preferably all residues of these sequences. As usedherein the term Flavivirus inhibitory peptides preferably means peptideshaving a sequence identical to the corresponding portion of theFlavivirus inhibitory protein and peptides in which one or more aminoacids are substituted by functionally equivalent amino acids (seeinfra). The term also refers to derivatives of these peptides, includingbut not limited to benzylated derivatives, glycosylated derivatives, andpeptides which include enantiomers of naturally occurring amino acids.In other embodiments of the invention, the Flavivirus inhibitorypeptides, related peptides or derivatives are linked to a carriermolecule such as a protein. Proteins contemplated as being usefulaccording to this embodiment of the invention, include but are notlimited to, (human serum albumen). Flavivirus inhibitory peptide-relatedpeptides comprising additional amino acids are also contemplated asuseful according to the invention.

Peptides may be produced from naturally occurring or recombinant viralproteins, or may be produced using standard recombinant DNA techniques(e.g. the expression of peptide by a microorganism which containsrecombinant nucleic acid molecule encoding the desired peptide, underthe control of a suitable transcriptional promoter, and the harvestingof desired peptide from said microorganism). Preferably, the peptides ofthe invention may be synthesized using any methodology known in the art,including but not limited to, Merrifield solid phase synthesis(Clark-Lewis et al., 1986, Science 231:134-139).

The FIP, or fragments or derivatives thereof, of the invention include,but are not limited to, those containing, as a primary amino acidsequences the amino acid sequence HCV Fusion Inhibitory Protein 1,X-YQVRNSSGLYHVTNDCPNSSIVYEAADAIL-Z (SEQ ID NO:1); HCV Fusion InhibitoryProtein 2 X-CSALYWVGDLCGSVFLVGQLFTFSPRRHWTTQDC-Z (SEQ ID NO:2); HCVFusion Inhibitory Protein 3, X-SPRRHWTTQDCNCSIYPGHITGHRMAWDMMNWSPT-Z(SEQ ID NO:3); or HCV Fusion Inhibitory Protein 4,X-MMMNWSPTAALLRIPQAIMDMIAGAHWGVLAGIKYFSMVGNW-Z (SEQ ID NO:4), or afunctional portion or functional portions thereof. Also contemplated arehomologous peptide sequences associated with another Flaviviruses,including, but not limited to, HGB, DV, JEV, YFV, WNV, CSFV, BVDV, orBDV. Also contemplated are altered sequences (i.e. altered from any ofthe sequences referred to herein) in which functionally equivalent aminoacid residues are substituted for residues within the sequence,resulting in a functionally silent change. For example, one or moreamino acid residues within the sequence can be substituted by replacingthe original amino acid with another amino acid, of a similar polarity,that acts as a functional equivalent, resulting in a functionally silentalteration. Substitutes for an amino acid within the sequence may beselected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. For example, and not by way of limitation, such peptidesmay also comprise one or more D-amino acids. Furthermore, in any of theembodiments of the instant invention the peptide may comprise aninefficient carrier protein, or no carrier protein at all.

5.3. Utility of the Invention

The Flavivirus inhibitory peptides of the instant invention may beutilized to inhibit Flavivirus virion:cell fusion and may, accordingly,be used in the treatment of Flavivirus infection and also in prophylaxisagainst Flavivirus infection. The peptides of the invention may beadministered to patients in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. Methods for administering peptides to patients arewell known to those of skill in the art; they include, but are notlimited to, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, oral, and intranasal. In addition, it may be desirable tointroduce the pharmaceutical compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection.

The instant invention provides for pharmaceutical compositionscomprising Flavivirus inhibitory peptides, peptide fragments, orderivatives (as described supra) administered via liposomes,microparticles, or microcapsules. Various embodiments of the invention,contemplate the use of such compositions to achieve sustained release ofFlavivirus inhibitory peptides. Other embodiments contemplate theadministration of the FIP or derivatives thereof, linked to a molecularcarrier (e.g. HSA).

Various embodiments of the instant invention provide for administrationof the Flavivirus inhibitory peptides and/or antibodies specific for thethese peptides to human or animal subjects who suffer from Flavivirusinfection (e.g. dengue hemorrhagic fever, West Nile disease, hepatitis Cor classical swine fever). In any embodiment the peptides and/orantibodies are typically substantially purified (as used herein the term“substantially purified” refers to a peptide, peptide analog, orantibody that is greater than about 80% pure. More preferably,“substantially purified” refers to a peptide, peptide analog, orantibody that is greater than about 90% or greater than about 95% pure.Most preferably it refers to a peptide, peptide analog, or antibody thatis greater than 99% pure. Functionally, “substantially purified” meansthat it is free from contaminants to a degree that that makes itsuitable for the purposes provided herein. Other embodiments provide forthe prophylactic administration of the peptides to those at risk forFlavivirus infection.

Other embodiments of the instant invention provide for methods foridentifying the structure of truncated Flavivirus fusion proteins whichinvolved in virion:cell fusion by members of the Flaviviridae family andfor the structures themselves.

Other embodiments of the invention provide for a peptide having aformula selected from one or more of the following.

A. Various embodiments of the invention provide for hepatitis C virusFusion Inhibitory Peptides: hepatitis C virus Fusion Inhibitory Protein1, X-YQVRNSSGLYHVTNDCPNSSIVYEAADAIL-Z (SEQ ID NO:1); HCV FusionInhibitory Protein 2, X-CSALYWVGDLCGSVFLVGQLFTFSPRRHWTFQDC-Z (SEQ IDNO:2); HCV Fusion Inhibitory Protein 3,X-SPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPT-Z (SEQ ID NO:3); or HCV FusionInhibitory Protein 4, X-MMMNWSPTAALLRIPQAIMDMIAGAHWGVLAGIKYFSMVGNW-Z(SEQ ID NO:4)

B. Other embodiments of the invention provide for a peptide or peptidehomolog wherein. the Flavivirus is member or tentative member of thehepacivirus genus. A preferred embodiment of this invention is drawn toa peptide or peptide analog wherein the tentative member of thehepacivirus genus is hepatitis G virus and peptides are selected fromthe group consisting of: hepatitis G virus Fusion Inhibitory Peptides:hepatitis G virus Fusion Inhibitory Protein 1,X-RVTDPDTNTTILTNCCQRNQVIYCSPSTCL-Z (SEQ ID NO:5); hepatitis G virusFusion Inhibitory Protein 2, X-TCDALDIGELCGACVLVGDWLVRHWLIHIDLNET-Z (SEQID NO:13); hepatitis G virus Fusion Inhibitory Protein 3,X-IHIDLNETGTCYLEVPTGIDPGFLGFIGWMAGKVEA-Z (SEQ ID NO:21); or hepatitis Gvirus Fusion Inhibitory Protein 4,X-WMAGKVEAVIFLTKLASQVPYAIATMFSSVHYLAVGALIYYS-Z (SEQ ID NO:29)

C. Other embodiments of the invention provide for a peptide or peptidehomolog from the flavivirus genus. In a preferred aspect of thisembodiment, the peptide or peptide analog is from dengue virus and thepeptides are selected from the group consisting of dengue virus FusionInhibitory Peptides: dengue virus Fusion Inhibitory Protein 1,X-RDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDF-Z (SEQ ID NO:6); dengue virusFusion Inhibitory Protein 2, X-KRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTC-Z (SEQID NO:14); dengue virus Fusion Inhibitory Protein 3,X-MVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNW-Z (SEQ ID NO:22); or dengue virusFusion Inhibitory Protein 4,X-MVLLGDTAWDFGSLGGVFFSIGKALHQVFGAIYGAAFSGVSW-Z (SEQ ID NO:30).

D. Other embodiments of the invention provide for peptides or peptidehomolog from flavivirus genus member, Japanese encephalitis virus. Inpreferred aspects of these embodiments the peptides and or/peptideanalogs are selected from the group consisting of: Japanese encephalitisvirus Fusion Inhibitory Peptides: Japanese encephalitis virus FusionInhibitory Protein 1, X-RDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDV-Z (SEQ IDNO:7); Japanese encephalitis virus Fusion Inhibitory Protein 2,X-SSYVCKQGFTDRGWGNGCGLFGKGSIDTCAKFSC-Z (SEQ ID NO:15); Japaneseencephalitis virus Fusion Inhibitory Protein 3,X-FYVMTVGSKSFLVHREWFHDLALPWTSPSSTAWRNR-Z (SEQ ID NO:23); or Japaneseencephalitis virus Fusion Inhibitory Protein 4,X-LAALGDTAWDFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMSW-Z (SEQ ID NO:31).

E. Other embodiments of the invention provide for peptides and/orpeptide homologs wherein the member of the flavivirus genus is yellowfever virus and the peptides are selected from the group consisting of:yellow fever virus Fusion Inhibitory Peptides: yellow fever virus FusionInhibitory Protein 1, X-RDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDI-Z (SEQ IDNO:8); yellow fever virus Fusion Inhibitory Protein 2,X-GDNACKRTYSDRGWGNGCGLFGKGSIVACAKFTC-Z (SEQ ID NO:16); yellow fevervirus Fusion Inhibitory Protein 3,X-SYIAEMETESWIVDRQWAQDLTLPWQSGSGGVWREM-Z (SEQ ID NO:24); or yellow fevervirus Fusion Inhibitory Protein 4,X-LAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNW-Z (SEQ ID NO:32).

F. Other embodiments of the invention provide for peptides and/orpeptide homologs of wherein the member of the flavivirus genus is WestNile virus and the peptides are selected from the group consisting of:West Nile virus Fusion Inhibitory Peptides: West Nile virus FusionInhibitory Protein 1, X-RDFLEGVSGATWDLVLEGDSCVTIMSKDKPTIDV-Z (SEQ IDNO:9); West Nile virus Fusion Inhibitory Protein 2,X-PAFVCRQGVVDRGWGNGCGLFGKGSIDTCAKFAC-Z (SEQ ID NO:17); West Nile virusFusion Inhibitory Protein 3, X-YYVMTVGTKTFLVHREWFMDLNLPWSSAGSTVWRNR-Z(SEQ ID NO:25); or West Nile virus Fusion Inhibitory Protein 4,X-LAALGDTAWDFGSVGGVFTSVGKAVHQVFGGAFRSLFGGMSW-Z (SEQ ID NO:33).

G. Other embodiments of the instant invention provide for peptidesand/or peptide homologs wherein the Flavivirus is a member of thepestivirus genus. In various aspects of these embodiments he peptides orhomologs thereof the member of the pestivirus genus is classical swinefever virus and the peptides are selected from the group consisting of:classical swine fever virus Fusion Inhibitory Peptides: classical swinefever virus Fusion Inhibitory Protein 1,X-GQLACKEDYRYAISSTNEIGLLGAGGLTTTWKEYN-Z (SEQ ID NO:10); classical swinefever virus Fusion Inhibitory Protein 2,X-KGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPT-Z (SEQ ID NO:18); or classicalswine fever virus Fusion Inhibitory Protein 3,X-TLRTEVVKTFRRDKPFPHRMDAVTTWKENEDLFY-Z (SEQ ID NO:26); or classicalswine fever virus Fusion Inhibitory Protein 4,X-QQYMLKGEYQYWFDLDVTDRHSDYFAEFVVLVVVALLGGRYI-Z (SEQ ID NO:34).

H. Other embodiments of the instant invention provide for peptides andpeptide homologs wherein the member of the pestivirus genus is bovineviral diarrhea virus and the peptides are selected from the groupconsisting of: bovine viral diarrhea virus Fusion Inhibitory Peptides:bovine viral diarrhea virus Fusion Inhibitory Protein 1,X-GHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYS-Z (SEQ ID NO:11); bovine viraldiarrhea virus Fusion Inhibitory Protein 2,X-RGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMD-Z (SEQ ID NO:19); or bovine viraldiarrhea virus Fusion Inhibitory Protein 3,X-TLATEVVKIYKRTKRFRSGLVATHTTIYEEDLYH-Z (SEQ ID NO:27); or bovinediarrhea virus Fusion Inhibitory Peptide 4,X-QQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYV-Z (SEQ ID NO:35).

I. Other embodiments of the instant invention provide for peptides andpeptide homologs wherein the member of the pestivirus genus is borderdisease virus and the peptides are selected from the group consistingof: border disease virus Fusion Inhibitory Peptides: classical swinefever virus Fusion Inhibitory Protein 1,X-GEFACREDHRYALAKTKEIGPLGAESLTTTWTDYQ-Z (SEQ ID NO:12); border diseasevirus Fusion Inhibitory Protein 2,X-RGKYNATLLNGSAFQLVCPYEWTGRVECTTISKS-Z (SEQ ID NO:20); or border diseasevirus Fusion Inhibitory Protein 3, X-TLATTVVRTYRRSKPFPHRQGAITQKNLGEDLH-Z(SEQ ID NO:28); or border disease virus Fusion Inhibitory Peptide 4,X-QQYMLKGQYQYWFDLEVISSTHQIDLTEFIMLAVVALLGGRYV-Z (SEQ ID NO:36)

In any of the foregoing groups the amino acids are represented by thesingle letter code. In various aspects of these embodiments “X”comprises an amino group, an acetyl group, a hydrophobic group or amacromolecular carrier group; “Z” comprises a carboxyl group, an amidogroup a hydrophobic group or a macromolecular carrier group. In otheraspects of this embodiment of the invention, X is a hydrophobic group, acarbobenzoxyl group, a dansyl group, t-butyloxycarbonyl group, a lipidconjugate, a polyethylene glycol group, or a carbohydrate. In any aspectof this embodiment Z may be a t-butyloxycarbonyl group, a lipidconjugate, a polyethylene glycol group, or a carbohydrate.

Moreover, aspects of this embodiment also include peptides wherein atleast one bond linking adjacent amino acids. residues is a non-peptidebond. In particularly preferred aspects of this embodiment thenon-peptide bond is an imido, ester, hydrazine, semicarbazoide or azobond.

Other aspects of this embodiment provide for peptides wherein at leastone amino acid is a D-isomer amino acid.

Additional aspects of this embodiment of the invention provide forpeptides wherein compromising at least one amino acid substitution hasbeen made so that a first amino acid residue is substituted for asecond, different amino acid residue. These substitutions may beconservative or non-conservative. So long as the peptide is stillfunctional according to the instant invention.

Other aspects of this embodiment of the invention provide for peptideswherein at least one amino acid has been deleted. As noted, supra, thepeptides according to this embodiment of the invention must comprise atleast 3 contiguous amino acids of one of the SEQ ID NOs indicated aboveand must be a functional segment.

It is noted that any combination of the modifications listed above isconsidered as part of the instant invention.

6. EXAMPLE HEPATITIS C VIRUS E1 IS A TRUNCATED CLASS II FUSION PROTEIN

Proteomics computational tools were used to fit HCV E1 protein to thescaffold of TBEV E, the prototypic class II fusion protein. Because HCVE1 is shorter than TBEV E, we reasoned that the former might containseveral “deletions” relative to the latter. The HCV E1 fusion peptide(Flint et al., 1999) was assumed to be located at the end of themolecule furthest from the carboxyl termiinal (C-terminal) transmembraneanchor domain, and, like other class II fusion proteins to be comprisedmostly of antiparallel β-sheets. This latter assumption was supported byChou-Fasman (Chou and Fasman, 1974) and Robson-Garnier (Biou et al.,1988) analyses, the most commonly applied secondary structure predictionalgorithms.

The fusion peptide of HCV (amino acids [aa] 272 to 281 of thefull-length polyprotein) was aligned with the fusion peptide of TBEV E(aa 385-396) (FIG. 1A). Both TBEV E and HCV E1 fusion peptides havecysteine residues at either end and contain a core of mostly aromaticand hydrophobic amino acids (FIG. 1A). Another domain readilyidentifiable in HCV E1 is the transmembrane domain. Amino acids 361 to381 of the hydrophobic sequence near the carboxyl terminus of E1 werepredicted to form a transmembrane helix by TMpred (transmembraneprediction software, see ch.embnet.org) (TMpred score 1308, >500 isstatistically significant).

Several regions of predicted β sheets and α helices in HCV E1 showedsimilarities to sequences known to assume those secondary structures inTBEV E (FIG. 1A). Beginning from the amino terminus, the firstsimilarity of HCV E1 begins in β sheet D_(o) of TBEV E and extendsthrough the fusion peptide. PRSS3, a sequence alignment algorithm, wasused to confirm that there is a significant similarity (p<0.025) betweenamino acids 246-281 of HCV E1 and amino acids 350-396 of TBEV E (FIG.1B). The fusion peptide is flanked by β sheets in class II fusionproteins and predicted β sheets with similarities to the b and c βsheets of TBEV E are indeed predicted to be present on either side ofthe putative HCV E1 fusion peptide by Chou-Fasman and Robson-Garnieranalysis. HCV E1 also has an extended region of similarity with theamino acid sequence between the two longest helices in TBEV E, αA andαB. There is a statistically significant (P<0.025) alignment of aminoacids 316-356 of HCV E1 with amino acids 496-544 of TBEV E (FIG. 1B).

To determine the plausibility of these alignments, a three-dimensionalmodel of HCV E1 was scaffolded on domain II of TBEV E (FIG. 2A). Similarsequences/structures were drawn in similar locations. Reorienting the“b” sheet in E1 is the only change relative to E required to bring theeight cysteine residues into close proximity. The four dicysteines ofHCV E1 potentially form a “zipper” down the center of the molecule likethe three dicysteines in domain II of TBEV E (FIG. 2B). This modellocates the five HCV E1 glycosylation sites so they are surfaceaccessible. Additionally, most of the hydrophobic residues are presentin a region on one side of E1 between the fusion peptide and thetransmembrane anchor (see below, FIG. 5).

Each of the HCV E1 structures drawn in FIG. 2B conforms to bothChou-Fasman and Robson-Garnier predictions, with the exception of theregion from “i” to “αB”. The structures designated “i” and “j” werepredicted to be β sheets by Chou-Fasman analysis, but α helical byRobson-Garnier analysis. The structure designated “αB” was predicted tobe a β sheet by Chou-Fasman analysis, but α helical by Robson-Garnieranalysis. HCV E1 appears to be missing, relative to TBEV E, much of theportion of the molecule prior to the transmembrane helix (pre-anchor).This region of TBEV E follows the trypsin cleavage site at amino acid395 used to generate that portion of the ectodomain of E examined byX-ray crystallography, and therefore, the TBEV E pre-anchor (stem)structure is uncertain. The pre-anchor of TBEV E has been predicted toform an amphipathic a helix (Allison et al., 1999). A sequence (aa693-721) of the pre-anchor domain in TBEV E has the characteristics of aleucine zipper, i.e. leucine or another hydrophobic amino acid in thefirst and fourth (a and d) positions of a seven amino acid periodicity(FIG. 1A). The pre-anchor sequence of HCV E1 was also predicted to be anα helix with characteristics of a “leucine zipper” (Charloteaux et al.,2002). Because of the significant amino acid sequence similarity withTBEV E, the HCV E1 secondary structures between “αA” and “αB” weredepicted as in TBEV E. There are several possible alternatives to the 3Dmodel of HCV E1 drawn in FIG. 2B, and it is possible that the secondarystructures change on interaction with membranes.

In contrast to HCV E1, our analyses did not reveal any sequences of HCVE2 with significant similarity to any sequence in domains I or II ofTBEV E or any other flavivirus E protein (representatives of each of thefour major serogroups were examined). Most of the N-terminal half of HCVE2, which include hypervariable region 1 (HVR 1), is without anysequence similarity to TBEV E. However, we detected a significantalignment (p<0.025) of the C-terminal half of HCV E2 (aa 549-726) withthe region of TBEV E (aa 590-763) from domain III through the first oftwo predicted transmembrane spanning domains of TBEV E (FIG. 1, TBEV ETM1, amino acids 448-469, TMpred: 1496; TM2, amino acids 474496, TMpred:1962). As discussed above, the pre-anchor region of TBEV E has asequence (aa 693-721) with features of a “leucine zipper; a similarmotif (aa 675-703) is found in the HCV E2 pre-anchor (FIG. 1). Inaddition, the carboxyl (C) terminus of HCV E2, like that of TBEV E,contains a stretch of hydrophobic amino acids that potentially couldspan the membrane twice. The transmembrane anchor(s) of HCV E2 (TMpredscore: 1364) is interrupted by charged amino acids like TM1 of TBEV E.Thus, by sequence alignments and structural predictions there aredemonstrable similarities between the C-terminal portions of HCV E2 andTBEV E.

Significant alignments of E1 of hepatitis GB virus (GBV-B) with HCV E1,indicate that this unclassified member of the Flaviviridae family alsoencodes a truncated class II fusion protein.

6.1. Materials and Methods

Prototype strains of representatives of the Flaviviridae were used forsequence and structural comparisons. The strains examined include TBEVstrain Neudoerfl (accession number: P14336); and the human prototypestrain H (subtype 1a) of hepatitis C virus (P27958), Some comparisonsused representatives of the major serogroups of flaviviruses, includingJapanese encephalitis virus, strain JaOARS982 (P32886), yellow fevervirus, strain 17D-204 (P19901), dengue virus type 2, strain PR-159/S1(P12823), and West Nile virus, strain NY 2000-crow3356 (AF404756). Wealso compared HCV sequences to those of GB virus-B virus (AAC54059), anunassigned member of the Flaviviridae.

MACMOLLY®, protein analysis software (Soft Gene GmbH, Berlin), was usedto locate areas of limited sequence similarity and to performChou-Fasman and Robson-Garnier analyses. PRSS3, a program derived fromrdf2 (Pearson and Lipman, 1988), which uses the Smith-Waterman sequencealignment algorithm (Smith and Waterman, 1981), was used to determinethe significance of protein alignments. PRSS3 is part of the FASTApackage of sequence analysis programs available by anonymous ftp fromftp.virginia.edu. Default settings for PRSS3 were used, including theblosum50 scoring matrix, gap opening penalty of 12, and gap extensionpenalty of 2. The alignments presented are those that produced thehighest alignment scores, rather than the longest sequences thatproduced significant scores. Chou-Fasman and Robson-Garnier algorithmspredict protein structures in an aqueous environment, but they cannotpredict protein structures in a lipid bilayer. Domains with significantpropensity to form transmembrane helices were identified with TMpred(ExPASy, Swiss Institute of Bioinformatics). TMpred is based on astatistical analysis of TMbase, a database of naturally occurringtransmembrane glycoproteins (Hofmann and Stoffel, 1993). RasMac,developed by Roger Sayle, was used to render 3D models of TBEV E.

6.2. Results and Discussion

The results indicate that the ectodomain of hepaciviruses is a truncatedversion of the class II fusion protein structure. The ectodomain of HCVE1 is roughly equivalent to the part of TBEV E from the “hinge” regionto the fusion peptide (FIG. 2). Our conclusions contrast with those ofYagnik et al. (2000), who predicted that HCV E2 fits the scaffold of acomplete class H fusion protein. These models were not previouslydescribed. Yagnik et al. (2000), taught that HCV E2 fits the scaffold ofa complete class II fusion protein. Lescar and co-workers (2001) statedthat their structural determinations of SFV E1, which established theexistence of a second class of fusion proteins, “indeed support theproposed model of the hepatitis C virus envelope protein E2 which wasbased on the 3D structure of the flavivirus envelope protein E.” Incontrast our model indicated that HCV E1 is class II although notsimilar to that previously described. Although there are sequence andstructural similarities between HCV E2 and TBEV E, these similaritiesare limited to the C-terminal portions of these proteins, and aredifferent than those proposed previously (Yagnik et al., 2000).

7. EXAMPLE PESTIVIRUS E2 IS A TRUNCATED CLASS II FUSION PROTEIN

To provide additional evidence for the HCV E1 class II fusion proteinmodel, we determined whether the fusion proteins of the thirdFlaviviridae genus, pestiviruses, might share structural/sequentialsimilarities with fusion proteins of members of the flavivirus andhepacivirus genera. Pestiviruses encode three envelope glycoproteins,Erns, E1 and E2. Erns, a secreted protein with RNAse activity, does nothave a hydrophobic transmembrane anchor domain. Erns does possess aC-terminal charged amphipathic segment that can mediate translocation ofErns across bilayer membranes (Langedijk, 2002). Pestivirus E1 and E2both have C-terminal hydrophobic domains that could function astransmembrane anchors. Therefore, we postulated that either pestivirusE1 or E2 must be the pestivirus fusion protein.

A putative fusion peptide (aa 818-828) is present in CSFV E2, containinga consensus sequence with aromatic and hydrophobic amino acids locatedbetween two cysteine residues (FIG. 1). The cysteine residues as well asthe sequences in between are highly conserved among pestiviruses, as istrue of fusion peptides from other enveloped RNA viruses of class I andII (not shown). Although statistically significant alignments were notdetected between the N-terminus of CSFV E2 and TBEV E (or between otherflaviviruses), a significant alignment (p<0.01) was detected betweenCSFV E2 (aa 792-835) and HCV E1 (aa 253-294) in this region (FIG. 1B).Furthermore, sequences flanking the putative fusion peptide werepredicted to form β sheets by both Chou-Fasman and Robson-Garnieranalyses (supplemental data). A significant alignment (p<0.05) betweenCSFV E2 (aa 841-913) and HCV E1 (aa 301-383) was also determined. Byextension, the central portion of CSFV E2 is predicted to structurallyresemble domain II of TBEV E. A significant alignment (p<0.005) wasdetected between amino acids 914-1018 of CSFV E2 and a sequence indomain III of TBEV E (aa 587-685) (FIG. 1B). There was also asignificant similarity (p<0.005) of this region of CSFV E2 (aa 914-1123)with a sequence (aa 549-743) in the region of HCV E2 that aligns withTBEV domain III. In addition, TMpred confirmed that the hydrophobicC-terminal domain of CSFV E2 has a high propensity to span the lipidbilayer (score: 1137). Like the transmembrane domains of HCV E1/E2 andTBEV TM1, the putative transmembrane anchor of CSFV E2 has a centralpositive charge.

On the basis of the regions of significant sequence similarities betweenCSFV E2, HCV E1/E2 and TBEV E, coupled with the internal location of apossible fusion peptide, we conclude that relative to TBEV E, CSFV E2 islacking a portion of domain I including segments corresponding to βsheets E_(o) through I_(o). CSFV E2 also appears to contain a somewhatshorter segment relative to TBEV E in the pre-anchor domain, i.e. thesequence between the alignment with TBEV E domain III and thetransmembrane domain (FIG. 1B). No leucine zipper is evident in thepre-anchor of CSFV E2. A three dimensional model of CSFV E2 (FIG. 2C)confirms that the alignment in FIG. 1 is plausible. Each of the cysteineresidues is in proximity to other cysteine residues and potentially formdicysteine bridges. Like HCV E1, CSFV E2 conforms to the structure of atruncated class II fusion protein, albeit with fewer truncationsrelative to flavivirus E than HCV E1. Because E2 is conserved among thepestivirus genus, the similarities of CSFV E2 with TBEV E extend toother pestiviruses.

None of the E1 envelope glycoproteins of any pestivirus bear anysignificant sequence similarities to any sequenced flavivirus E protein.Immature flavivirus virions contain a precursor, prM, to the smallmembrane protein M. prM is cleaved in the endoplasmic reticulum by furinor by a furin-like protease during virus release to produce the mature Mprotein localized on the surface of flavivirus virions (Stadler et al.,1997). A sequence (amino acids 173-256) of CSFV E1 has similarity(p=0.030) to amino acids 583654 of TBEV prM (FIG. 3A). CSFV E1 does notcontain the sequence RXR/KR (SEQ ID NO:37), the furin consensus cleavagesite. CSFV E1 also does not contain an identifiable fusion peptide,although TMpred predicts a significant transmembrane spanning domain inthe first third of CSFV E1. Like the transmembrane domains of TBEV E,HCV E1 and E2 and CSFV E2, and TBEV prM (TMpred score=1828), theC-terminus of CSFV E1 is predicted to form a membrane spanning domain(TMpred score=1884) with a central positive charge.

7.1. Materials And Methods

The Alfort 187 strain of classical swine fever virus, aka hog choleravirus (CAA61161) was used as the prototype of the pestivirus genus ofthe family Flaviviridae. Type species of other pestiviruses, includingbovine viral diarrhea virus (BVDV) genotype 1, aka pestivirus type 1,strain NADL (CAB91847) and border disease virus strain BD31 (AAB37578),were used in other comparisons. Proteomics computational methods were asdescribed in 6.1.

7.2. Results And Discussion

Pestivirus E2 proteins are truncated class II fusion proteins, althoughwith fewer truncations relative to flavivirus E than hepacivirus E1.

8. EXAMPLE GENE ORDER OF FLAVIVIRIDAE GENOMES

Genes that encode proteins with similar functions may be present insimilar locations in genomes of different members of the Flaviviridaefamily. The positive-polarity single-stranded RNA genomes of all membersof the Flaviviridae are translated into a single large polyprotein thatis subsequently cleaved by viral and cellular proteases into functionalproteins. The order (from N to C terminus) of proteins in thepolyproteins of TBEV and other members of the flavivirus genus isC-prM-E-nonstructurals (C: capsid), and the order of proteins in thepolyproteins of hepaciviruses is C-E1-E2-p7-nonstructurals (FIG. 4). The5′ portion of the flavivirus E gene encodes the fusion peptide in domainII of the E protein, whereas the receptor binding domain of E isprobably located in domain III encoded by the 3′ portion of the E gene(Crill and Roehrig, 2001; Mandl et al., 2000). Fusion and receptorfunctions may reside in two different HCV proteins, E1 and E2respectively, occurring in the same order as the domains of flavivirus Ethat carry out these functions (FIG. 4). Hepacivirus E1 and E2 may havearisen by insertion of a transmembrane anchor and variable domains,including hypervariable region 1 (HVR-1, FIG. 1), into the ancestral Egene. Alternatively, HCV E1 could have evolved into a separate fusionprotein from an ancestral prM, with concurrent lost of the fusionpeptide and fusion functions in E2. The sequence similarities betweenTBEV E and HCV E1 and E2, however, do not favor this latter possibility.

The order of proteins in pestivirus polyproteins isNpro-C-Erns-E1-E2-p7-nonstructurals. Pestiviruses encode two proteins,Npro and E^(ms), with no obvious homologs among members of the other twoFlaviviridae genera. Pestivirus E1 and E2 are similar in sequence toflavivirus M and E, respectively. Like TBEV E, pestivirus E2 may serveboth as fusion protein and receptor binding protein. These functions arecarried out by TBEV E domains II and III that appear to be representedby similar structures in pestivirus E2 (FIG. 4). TBEV PrM/M functions toprotect internal cellular membranes from fusion mediated by E2, and itis possible that pestivirus E1 serves the same function for E2, thefusion/receptor protein. Excepting Npro and E^(ms), the order ofstructural proteins with sequence and other similarities is analogous inpestiviruses and flavivirus polyproteins.

TBEV E has two hydrophobic C-terminal transmembrane domains, TM1 and TM2(FIG. 1). Hepaciviruses and pestiviruses encode a small hydrophobicpeptide, “p7”, which could associate with cellular or viral membranes.The cleavage that produces p7 is inefficient and delayed, and thereforemuch of HCV E2 and pestivirus E2 are present in the cell as uncleavedE2-p7 precursors (Harada, Tautz, and Thiel, 2000). The p7 gene islocated in a similar genomic location and could have evolved from thesequence encoding the second transmembrane domain, TM2, of flavivirus E(FIG. 4). The consensus Flaviviridae genome can therefore be representedas X1—C—X2-M-fusion-binding-TM1-TM2-nonstructurals-3′, where X 1 and X2represents inserted sequences in pestiviruses, N^(pro) and E^(rms),respectively, M represents flavivirus prM/M-pestivirus E1 and TM2 is thesecond transmembrane domain of flaviviruses and p7 of hepaciviruses andpestiviruses. These similarities in gene order and functions support thehypothesis that E1 is the fusion protein of HCV.

8.1. Materials And Methods

Prototype strains of representatives of the Flaviviridae as described in6.1 and 7.1 were used for sequence comparisons.

8.2. Results And Discussion

Hepaciviruses, like alphaviruses, appear to use one envelope protein forattachment (E2) and another for fusion (E1). In contrast, Eglycoproteins of TBEV, dengue virus, and other members of the flavivirusgenus mediate both receptor binding and membrane fusion functions. E2functions as one of the pestivirus receptor-binding protein (Hulst andMoormann, 1997), and if the current analysis is correct also carries outthe virion:cell fusion function. In addition to E, flaviviruses encode amembrane protein prM whose functions may include shielding of cellularmembranes from the fusion peptide of E (Kuhn et al., 2002). Functions ofthe flavivirus small membrane protein may be vested in E1 ofpestiviruses, which has significant sequence similarity with flavivirusprM. Mature flavivirus virions contain prM that has been cleaved to M.Unlike M, pestivirus E1 does not associate with the virion envelope as aprecursor protein and lacks a furin cleavage site.

The Flavivirus fusion protein structures and functional domainsdescribed here are supported by the observations that envelopeglycoproteins with significant sequence similarities, HCV E1/2, TBEV Eand pestivirus E2 and TBEV prM and pestivirus E1 are in analogouslocations in the polyproteins encoded by the three genera of theFlaviviridae. These results suggest that members of the Flavivirusfamily may have a common ancestor. Divergence of the genes for thefusion proteins within the three genera of this family may have occurredeither through acquisition of sequences and/or lose of sequences in acassette manner constrained by the domain organization of class IIfusion proteins.

9. EXAMPLE MEMBRANE INTERFACIAL DOMAINS IN A CLASS I FUSION PROTEIN ANDHCV E1

Although the overall structures of class I and II fusion proteins aredistinct, they may share structural/functional characteristics in theparts of the molecules that interact with and disrupt bilayer membranes.It is well established that class I fusion proteins have a fusionpeptide at the amino terminus of the molecule that is critical forfusion (Gallaher, 1987; Gallaher, 1996; Gallaher et al., 1989; Gallaher,DiSimone, and Buchrneier, 2001). Class II fusion proteins have aninternal fusion peptide that are located after secondary structuralfolding at distal locations from the transmembrane anchor (Kuhn et al.,2002; Lescar et al., 2001; Rey et al., 1995). To provide further supportfor the proposed models of HCV E1 and pestivirus E2, we used anotherproteomics computational tool to compare other potential membraneinteractive domains in the proteins with the HIV-1 transmembraneglycoprotein (M), a class I fusion protein. Besides fusion peptides,another motif in class I fusion proteins that can be important invirus:cell fusion is an aromatic amino acid rich motif proximal to theanchor (FIG. 5A, amino acids 667-683) (Suarez et al., 2000). Thepre-anchor domains of class I fusion proteins are not highly hydrophobicaccording to the Kyte-Doolittle hydropathy prediction algorithm,however, these domains have a tendency to partition into bilayermembranes, as revealed by analyses using the Wimley-White interfacialhydrophobicity scale (Suarez et al., 2000; Wimley and White, 1996). HCVE1 contains three domains that produce significant Wimley-Whitepartition scores using Membrane Protein explorer (Jaysinghe, Hristova,and White, 2000). One of these is the transmembrane anchor (aa 361-372).The other two sequences with significant Wimley-White partition scoresare located immediately following the fusion peptide (aa 284-300) and ata location (aa 321-340) that the model in FIG. 2B predicts to be nearthe bilayer membrane (FIG. 5B).

9.1. Materials And Methods

Sequences with a propensity to partition into the lipid bilayer wereidentified with Membrane Protein explorer from the Stephen Whitelaboratory (Jaysinghe, Hristova, and White, 2000) using defaultsettings.

9.2. Results And Discussion

These two HCV E1 domains, in conjunction with the fusion peptide and thetransmembrane anchor, potentially form a continuous track of membraneinteractive regions that could channel the movement of lipids duringvirion:cell fusion. These Wimley-White partition analyses thus provideadditional support for the proposal that E1 is the fusion protein ofHCV.

10. EXAMPLE IDENTIFICATION OF PEPTIDES THAT INHIBIT FUSION/INFECTIVITYMEDIATED BY HCV ENVELOPE PROTEINS

The membrane fusogenic envelope glycoproteins of Flaviviruses shareseveral common structural features, including “fusion peptides” andglobular domain structures consisting mostly of antiparallel β sheets.Furthermore, the E1 protein of HCV and the E proteins of DEN, WNV andYFV each have several motifs with a high propensity to interact withbilayer membranes as revealed by algorithms employing the Wimley-Whiteinterfacial hydrophobicity scale. These structural features and membraneinterfacial motifs are presumably important in Flavivirus fusion, entryand infection and may represent targets to develop peptide drugs againstFlavivirus infection.

10.1. Materials And Methods

To overcome the lack of a conventional cell culture system for thepropagation of HCV, infectious pseudotype viruses expressing HCVenvelope glycoproteins have been generated (Hsu et al., 2003).Pseudotypes with HIV core proteins and HCV envelope proteins weregenerated by cotransfection of 293-T cells with equal amounts ofplasmids expressing HCV E1 and E2 of strain H77 and the HIVenvelope-defective proviral genome, pNL4.3.Luc.R⁻E⁻ (Pohlmann et al.,2003). Peptides from an 18mer peptide set, overlapping by 7-10 aminoacids and representing the entire amino acid sequence of E1 of HCVstrain H77, were solubilized in 20% DMSO, diluted (final DMSOconcentration <2%). Peptides were incubated on ice for 30 minutes withp24 antigen-normalized HCV pseudotype viral supernatants. The averageconcentration of peptides was ˜25 μM, however, actual concentrations ofsome peptides in solution were 10 μM or less due to low solubility inDMSO (marked by asterisk in Table 5). Supernatants were also treatedwith DMSO vehicle alone or with a Mab (monoclonal antibody) to HCV E2known to neutralize pseudotype infectivity. HCV peptides, vehicle, andanti-E2 MAb were also incubated with pseudotypes expressing murineleukemia virus (MLV) envelope proteins and HIV capsid proteins tocontrol for cytotoxicity. Peptide treated and control HCV and MLVpseudotypes were added to cells, which were incubated at 37° C. for 72h. Cell lysates were then tested for luciferase activity as described(Hsu et al., 2003).

10.2 Results and Discussion

Four HCV E1 peptides demonstrated greater than 70% inhibition of HCVpseudotype infectivity, with one (peptide 54) reducing HCV pseudotypeinfectivity by >99.9% (Table 5, FIG. 5B). Two of the peptides (66 and70) correspond to sequences with a high propensity to interact with thesurface of bilayer membranes, as determined by application of theWimley-White interfacial hydrophobicity scale. Peptide 66 also inhibitedinfection by the HIV(MLV) pseudotype by greater that 50% suggestingeither that this peptide is a general inhibitor of viral fusion or thatit is cytotoxic. The other two inhibitory peptides (54 and 74) representsequences of HCV E1 predicted to “fold” over and interact with theportion of E1 displaying high Wimley-Whitr interfacial hydrophobicityscores (FIG. 5B). The postulated folding over of these domains wasmarked by arrows in the original published figure (FIG. 5 of Garry andDash, 2003,). These results demonstrate the potential of peptides asantiHCV drugs, and indicate that similar strategies can identifypeptides that inhibit fusion and infectivity of other Flaviviruses.TABLE 5 Identification of lead peptides that inhibit infectivitymediated by HCV envelope proteins. Peptide Percent Percent number^(‡)H77-E1E2† inhibition ^(§)MLV† inhibition 52 133,259 −17.16 533,179−21.4 53 113,469 0.23 443,528 −9.95 54 74 99.93 280,113 36.22 55 112,4701.12 447,957 −2.00 56 65,612 42.32 433,459 1.30 57 169,860 −49.35331,852 24.44 58 118,767 −4.42 329,895 24.98 59 91,794 19.29 446,063−1.57 60 98,766 13.16 340,384 22.49 61 148,796 −30.83 423,925 3.47 62115,966 −1.96 415,014 5.50 63 57,915 49.08 438,440 0.16 64 113,108 0.55316,948 27.83 65* 87,726 22.87 491,789 −11.98 66 23,387 79.46 189,68356.81 67 64,601 43.20 357,577 28.58 68 79,297 31.28 498,991 −13.62 69*196,922 −73.14 354,027 19.39 70 15,717 86.19 553,120 −25.95 71 83,48926.60 533,765 −21.54 72 75,763 33.39 392,680 10.58 73 100,666 11.49433,001 1.40 74 32,888 71.09 467,876 −6.54 75 113,359 0.32 420,026 4.3676 96,283 15.34 473,757 −7.88 77 56,425 50.39 321,076 26.89 78* 137,700−21.07 402,953 8.24 79 101,702 10.58 740,034 −68.51 Vehicle 113,733 —439,158 — anti-E2 73 99.93 349,113 21.50^(‡)H77-E1E2 is the pseudotype expressing envelope glycoproteins E1 andE2 of the H77 strain of HCV.^(§)MLV is a similar pseudotype expressing the envelope glycoprotein ofmurine leukemia virus and serves as a peptide control.†The numbers represent the number of luciferase units (lumens) producedafter infection by either the HCV or the MLV pseudotype in the presenceof the peptide at a concentration of ˜25 μM.

TABLE 6 Sequence and Location of peptides shown in Table 5. PeptidePeptide Number Location Amino acid sequence FIP overlap 52 183-200SCLTVPASAYQVRNSSGL (SEQ ID NO:38) 53 190-207 SAYQVRNSSGLYHVTNDC (SEQ IDNO:39) HCV E1 FIP1 54 197-214 SSGLYHVTNDCPNSSIVY (SEQ ID NO:40) HCV E1FIP1 55 204-221 TNDCPNSSVVYEAADAIL (SEQ ID NO:41) HCV E1 FIP1 56 211-228SIVYEAADAILHTPGCVP (SEQ ID NO:42) 57 218-235 DAILHTPGCVPCVREGNA (SEQ IDNO:43) 58 225-242 GCVPCVREGNASRCWVAV (SEQ ID NO:44) 59 232-249WVAVTPTVATRDGKLPTT (SEQ ID NO:45) 60 239-256 WVAVTPTVATRDGKLPTT (SEQ IDNO:46) 61 246-263 VATRDGKLPTTQLRRHID (SEQ ID NO:47) 62 253-270LPTTQLRRHIDLLVGSAT (SEQ ID NO:48) 63 260-277 RHIDLLVGSATLCSALYV (SEQ IDNO:49) 64 267-284 GSATLCSALYVGDLCGSV (SEQ ID NO:50) HCV E1 FIP2 65274-291 ALYVGDLCGSVFLVGQLF (SEQ ID NO:51) HCV E1 FIP2 66 281-298CGSVFLVGQLFTFSPRHH (SEQ ID NO:52) HCV E1 FIP2/3 67 288-305GQLFTFSPRHHWTTQDCN (SEQ ID NO:53) HCV E1 FIP3 68 295-312PRHHWTTQDCNCSIYPGH (SEQ ID NO:54) HCV E1 FIP3 69 302-319QDCNCSIYPGHITGHRMA (SEQ ID NO:55) HCV E1 FIP3 70 309-326YPGHITGHRMANMMMNW (SEQ ID NO:56) HCV E1 FIP3/4 71 316-333HRMANMMMNWSPTAALV (SEQ ID NO:57) HCV E1 FIP3/4 72 323-340MMNWDPTAALVVAQLLRI (SEQ ID NO:58) HCV E1 FIP4 73 330-347AALVVAQLLRIPQAIMDM (SEQ ID NO:59) HCV E1 FIP4 74 337-354LLRIPQAIMDMAIGAHWG (SEQ ID NO:60) HCV E1 FIP4 75 344-361IMDMIAGAHWGVLAGIKY (SEQ ID NO:61) HCV E1 FIP4 76 351-368AHWGVLAGIKYFSMVGNW (SEQ ID NO:62) HCV E1 FIP4 77 359-375GIKYFSMVGNWAKVLVVL (SEQ ID NO:63) 78 365-382 VGNWAKVLVVLLLFAGVD (SEQ IDNO:64) 79 372-389 LVVLLLFAGVDAETHVTG (SEQ ID NO:65)

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of eachof which is incorporated by reference in its entirety. Citation oridentification of any reference in any section of this application shallnot be construed as an admission that such reference is available asprior art to the present invention.

REFERENCES

Each of the following is herein incorporated by reference in itsentirety.

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1. A pharmaceutical composition comprising one or more peptides selectedfrom the group consisting of: a) a peptide having the sequence of any ofSEQ ID NO:1 to SEQ ID NO:36; b) a peptide homologous to any one of SEQID NO:1 to SEQ ID NO:36 from another flavivirus; and c) a peptidefunctionally equivalent to any one of SEQ ID NO:1 to SEQ ID NO:36,wherein the functionally equivalent peptide is identical to at least oneof SEQ ID NO:1 to SEQ ID NO:36 except that one or more amino acidresidues has been substituted with a homologous amino acid, resulting ina functionally silent change, or one or more amino acids has beendeleted.
 2. A pharmaceutical composition comprising at least one peptideselected from the one or more of the following: a) a peptide having theamino acid sequence one or more of SEQ ID NO:1 to SEQ ID NO:36, whereinthe N-terminal amino acid residue comprises an N-terminal amino groupand the C-terminal amino acid residue comprises a c-terminal carboxylgroup; b) a peptide having the sequence of any of SEQ ID NO:1 to SEQ IDNO:36, wherein the chemical moiety at the peptide's N-terminus not anamino group and/or the chemical moiety at the peptide's C-terminus isnot a carboxyl group, wherein the N-terminal chemical moiety is selectedfrom the group consisting of: an acetyl group, a hydrophobic group,carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or amacromolecular carrier group, and/or wherein the C-terminal chemicalmoiety is selected from the group consisting of an amido group, ahydrophobic group, t-butyloxycarbonyl group or a macromolecular group;c) a peptide having the sequence of any of SEQ ID NO:1 to SEQ ID NO:36,wherein at least one bond linking adjacent amino acid residues is anon-peptide bond; d) a peptide having the sequence of any of SEQ ID NO:1to SEQ ID NO:36, wherein at least one amino acid residue is in theD-isomer configuration; e) a peptide as in part “a)” or “b)” except thatat least one amino acid has been substituted for by a different aminoacid; or f) a functional fragment of a peptide as set out in any ofparts “a)” to “e)”, having at least 3 contiguous nucleotides of any oneof SEQ ID NO:1 to SEQ ID NO:36.
 3. The composition of claim 2 whereinthe peptide is selected from one or more of the group consisting of SEQID NO:1, 2, 3, and
 4. 4. The composition of claim 3 wherein theN-terminal chemical moiety is an acetyl group, a hydrophobic group acarbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, or amacromolecular carrier group; and/or the C-terminal chemical moiety is ahydrophobic group, a t-butyloxycarbonyl group or a macromolecular group.5. The composition of claim 3 wherein the N-terminal chemical moiety isa macromolecular carrier group selected from a lipid conjugate,polyethylene glycol, or a carbohydrate; and/or the C-terminal chemicalmoiety is a macromolecular carrier group selected from a lipidconjugate, polyethylene glycol, or a carbohydrate.
 6. The composition ofclaim 3 wherein at least one bond linking adjacent amino acid residuesin the peptide is a non-peptide bond selected from the group consistingof an imido bond, an ester bond, a hydrazine bond, a semicarbazoide bondand an azo bond.
 7. The composition of 3 wherein at least one amino acidis a D-isomer amino acid.
 8. The composition of claim 3 whereinN-terminal chemical moiety is an amino group and the C-terminal chemicalmoiety is a carboxyl group.
 9. The composition of claim 2 wherein thepeptide is selected from one or more of the group consisting of SEQ IDNO:5, 13, 21, and
 29. 10. The composition of claim 9 wherein theN-terminal chemical moiety is an acetyl group, a hydrophobic group acarbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, or amacromolecular carrier group; and/or the C-terminal chemical moiety is ahydrophobic group, a t-butyloxycarbonyl group or a macromolecular group.11. The composition of claim 9 wherein the N-terminal chemical moiety isa macromolecular carrier group selected from a lipid conjugate,polyethylene glycol, or a carbohydrate; and/or the C-terminal chemicalmoiety is a macromolecular carrier group selected from a lipidconjugate, polyethylene glycol, or a carbohydrate.
 12. The compositionof claim 9 wherein at least one bond linking adjacent amino acidresidues in the peptide is a non-peptide bond selected from the groupconsisting of an imido bond, an ester bond, a hydrazine bond, asemicarbazoide bond and an azo bond.
 13. The composition of claim 9wherein at least one amino acid is a D-isomer amino acid.
 14. Thecomposition of claim 9 wherein the N-terminal chemical moiety is anamino group and the C-terminal chemical moiety is a carboxyl group. 15.The composition of claim 2 wherein the peptide is selected from one ormore of the group consisting of SEQ ID NO:6-9, 14-17, 22-25, and 30-33.16. The composition of claim 15 wherein the N-terminal chemical moietyis an acetyl group, a hydrophobic group a carbobenzoxyl group, a dansylgroup, a t-butyloxycarbonyl group, or a macromolecular carrier group;and/or the C-terminal chemical moiety is a hydrophobic group, at-butyloxycarbonyl group or a macromolecular group.
 17. The compositionof claim 15 wherein the N-terminal chemical moiety is a macromolecularcarrier group selected from a lipid conjugate, polyethylene glycol, or acarbohydrate; and/or the C-terminal chemical moiety is a macromolecularcarrier group selected from a lipid conjugate, polyethylene glycol, or acarbohydrate.
 18. The composition of claim 15 wherein at least one bondlinking adjacent amino acid residues in the peptide is a non-peptidebond selected from the group consisting of an imido bond, an ester bond,a hydrazine bond, a semicarbazoide bond and an azo bond.
 19. Thecomposition of claim 15 wherein at least one amino acid is a D-isomeramino acid.
 20. The composition of claim 15 wherein the N-terminalchemical moiety is an amino group and the C-terminal chemical moiety isa carboxyl group.
 21. The composition of claim 2 wherein the peptide isselected from one or more of the group consisting of SEQ ID NO: 10-12,18-20, 26-28, and 34-36.
 22. The composition of claim 21 wherein theN-terminal chemical moiety is an acetyl group, a hydrophobic group acarbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, or amacromolecular carrier group; and/or the C-terminal chemical moiety is ahydrophobic group, a t-butyloxycarbonyl group or a macromolecular group.23. The composition of claim 21 wherein the N-terminal chemical moietyis a macromolecular carrier group selected from a lipid conjugate,polyethylene glycol, or a carbohydrate; and/or the C-terminal chemicalmoiety is a macromolecular carrier group selected from a lipidconjugate, polyethylene glycol, or a carbohydrate.
 24. The compositionof claim 21 wherein at least one bond linking adjacent amino acidresidues in the peptide is a non-peptide bond selected from the groupconsisting of an imido bond, an ester bond, a hydrazine bond, asemicarbazoide bond and an azo bond.
 25. The composition of claim 21wherein at least one amino acid is a D-isomer amino acid.
 26. Thecomposition of claim 21 wherein the N-terminal chemical moiety is anamino group and the C-terminal chemical moiety is a carboxyl group. 27.A method of treating or preventing a Flavivirus infection comprisingadministering to the patient an effective amount of a pharmaceuticalcomposition according to claim
 1. 28. A method of treating or preventinga Flavivirus infection comprising administering to the patient aneffective amount of a pharmaceutical composition according to claim 2.29. A substantially purified antibody specific for a peptide asdescribed in claim
 1. 30. A substantially purified antibody specific fora peptide as described in claim 2.